Optical measurement systems are used for e.g. analysing properties or material contents of a target. Two basic prior art systems for optical measurement are next described with reference to FIGS. 1, 2a and 2b. FIG. 1 illustrates a system for measuring diffuse reflection of the target by using several wavelengths of light in order to measure contents of different substances in the target. A system with corresponding elements can also be used for measuring light absorbance of a target. FIGS. 2a and 2b illustrate time resolved measurement of fluorescence, whereby emitted light is received from a sample after excitation/activation with a light pulse. The emission of the target or its spectral reflection of ambient light like Sun light is detected using hyperspectral imaging spectrometers.
The system of FIG. 1 has a broadband light source 110, such as a halogen incandescent lamp. A light beam of the light source is collimated with lens 131 in order to lead the light beam through a filter wheel 121. The filter wheel has several filters 122a, 122b which each have a pass band of determined different wavelengths. Between the filters there is an area in the filter wheel which does not transmit light. When the filter wheel 121 rotates around its axis of rotation 123 each filter will successively enter the path of the light beam. Between entering any two filters there is a time period when the light beam of the light source is blocked by the filter wheel as the filter wheel may also serve as a shutter.
The filtered light beam of the light source is directed by a lens 133 into a suitable area at the surface of the target 150. Between the lens 133 and the target 150 there is a beam splitter mirror 135, which reflects a part of the light beam to lens 137 which focuses the beam to a reference detector 139. The reference detector 139 is used for providing feedback data about the intensity of the light beam in order to adjust the light intensity of the light source.
The light beam is reflected at the surface of the measurement target 150, and a part of the reflected light is focused with lenses 161 and 163 to a measurement detector 180. The detector 180 thus measures the intensity of the reflected light at each pass band wavelength of the filters 122a, 122b successively while the filter wheel 121 rotates. The time multiplexed signal received from the detector 180 thus includes the concentration information on corresponding substances in the mixture of the measurement target. The detector can be a point detector for achieving total intensity information of the light reflected from the illuminated area, or an image detector for achieving intensity information on spatial distribution of the reflected light within the illuminated area.
The system of FIG. 1 has certain disadvantages. The rotation speed of the filter wheel has a certain maximum value, and this causes that the time period between measurements with successive filters may be too long in some applications. An accurate measurement requiring repeated measurements also takes a relatively long time. There are also tight requirements for the similarity of the pass band filters needed for the performance of each instrument to be repeatable in terms of centre wavelengths and spectral resolutions at the selected bands. Such requirements cause the production costs of the filters to be high. There is also a problem of temperature dependence of the pass band centre wavelength of the interference filters. Therefore the temperature must be kept at a determined value with a strict tolerance.
The filter wheel is a relatively large component, which causes the measurement equipment to be large in size. Further, if the measurement wavelengths need to be changed, it is necessary to change the filter wheel. This is normally manual work and takes time. It may also be necessary to have a large number of filter wheels, which increases the investment on the equipment. The rotation of the filter wheel also requires an efficient motor, which consumes much energy. The filters are mechanically sensitive, so the rotation with high speed also includes a risk of damaging the filters.
Another solution has been described in prior art which solves a part of the above problems. This solution includes several detectors which each have an optical filter with determined pass band wavelengths. This way it is possible to avoid using a filter wheel. However, the narrowband filters in front of each detector are arranged in one plane but at different locations relative to the optical axis. Therefore the intensity distribution entering the detector plane should be homogenized in such a way that the spatial information from the target is not present in the intensity distribution entering the multiple detectors. Such accurate homogenization is difficult to achieve. Also, this solution does not solve the problems related to the temperature dependence of the filters nor the difficulty to change the pass band wavelengths to be measured.
Prior art hyperspectral imaging instruments are typically push-broom instruments based on Prism-Grating-Prism components, on aberration corrected holographic gratings or on linear variable filters. Push-broom instruments form a spectral image over one dimensional line at a time. This type of imaging spectrometers cannot produce 2D images fast because the target has to move or a scanning optics is required to form a 2D image.
There are prior art technologies which are capable of producing 2D dimensional spectral images by taking a 2D image of the target at predetermined wavelength of the measurement range. Such technologies include using Acousto-Optic Tunable Filters (AOTF) and Liquid Crystal Tunable Filters (LCTF). A typical wavelength-selective liquid crystal tunable filter is constructed from a stack of fixed filters consisting of interwoven birefringent crystal/liquid-crystal combinations and linear polarizers. The spectral region passed by LCTFs is dependent upon the choice of polarizers, optical coatings, and the liquid crystal characteristics (nematic, cholesteric, smectic, etc.). Both AOTF and LCTF technologies suffer from the low light transmission through the tunable filter. In LCTF technology the low transmission is caused by the fact that the light going through the filter must be polarized. In AOTF the light at the selected wavelength is diffracted to the direction of the detector but because the diffraction angle is rather small the throughput of the filter is limited.
FIG. 2a illustrates a system for the measurement of fluorescence from a sample which is located in a micro well 251. The system has a light source 210, which may be e.g. an UV LED or a Xenon flash tube. The light beam of the light source is collimated with a lens 231 and filtered with an excitation band filter 224. The excitation light beam is further focused with a lens 233 to the sample well via a beam splitter mirror 235 which reflects the main portion of the light beam. A minor part of the light beam transmits the mirror 235, and this part of the beam is focused to a reference detector 239, such as a photo diode. This reference detector 239 is used for monitoring the intensity of the excitation light pulses in order to provide feedback data for controlling the light intensity of the light source 210.
A direct fluorescence light is in this example emitted by Europium. An indirect FRET (Fluorescence Resonance Energy Transfer) light is emitted by Alexa647 fluorophore. The emission light from both fluorescence sources transmits the beam splitter 235, after which a dichroic beam splitter mirror 295 is used to separate the emission light into two channels; Alexa647 emission channel and Europium emission channel. The emission light beam from the Europium is reflected by the mirror 295 and collimated with a lens 291. The light beam is then filtered with a Europium emission band pass interference filter 292, which has pass band centre at e.g. 610 nm. The filtered light beam is then focused with a lens 293 to a detector 298, which is usually a photon counter, such as a photomultiplier tube.
The emission light beam from the Alexa647 fluorophore transmits the mirror 295 and it is then collimated with a lens 261. The light beam is then filtered with an Alexa647 emission band interference pass filter 272, which has pass band centre at e.g. 665 nm. The filtered light beam is then focused with a lens 263 to a detector 280, which is also usually a photon counter, such as a photomultiplier tube.
FIG. 2b shows a diagram of the fluorescence light 22 of the sample after the excitation light pulse has been exposed in a time resolved (TR) fluorescence measurement. The horizontal axis describes time and the vertical axis describes fluorescence intensity. After the excitation light pulse there is background fluorescence 26 for a short time period, and therefore the measurement window 24 is started after the background fluorescence has faded into a negligible intensity.
The system of FIG. 2a involves certain disadvantages. The FRET emission signals, such as Alexa647 emission signal, have a very small intensity, and therefore a sensitive photon counter is required. However, such photomultiplier tubes have a large size and they are expensive. On the other hand, semiconductor detectors such as CMOS and CCD image sensors are not sensitive enough for indirect TR-FRET measurements. When photomultiplier tube is used as a detector, it is only possible to measure one point/area of the target at a time, whereby providing an image as a measurement result requires a large number of measurements and moving the target or optics. When microtitration plates with several sample wells are measured, it is necessary to measure each well separately, which causes the measurements to take much time.
A further disadvantage of the prior art solutions described above is that it is difficult to optimize the spectrum of the measurement according to the target/measured substance of the target. While it is possible to produce filters with a specific pass band function, such filters are difficult to produce, and such a filter can be used for only one type of measurement/measured substance.